DIAGNÓSTICO

Oxidative stress occurs when there is an imbalance between the normal cellular production of pro-oxidants and endogenous antioxidant enzymes and plays a major role in the pathogenesis of numerous diseases such as CVD and risk factors thereof, such as ED (Ajuwon, Marnewick

& Davids, 2015).

Pro-oxidants are primarily composed of free radicals, defined as ions or atoms that have a non-paired electron on the outer orbital, thus rendered to be highly reactive. The highly reactive and unstable nature of the free radicals lead to generation of more free radicals. This occurs when they bind to macromolecules in search for electrons to complete the outer orbital, resulting to oxidation of the macromolecules. Sources of free radicals are; oxygen, nitrogen, chlorine and sulphur. When they react with oxygen and nitrogen, they form ROS and RNS, respectively.

ROS and RNS are essentially produced as by-products by aerobic organisms during normal metabolism, their examples are listed in Table 1.3 (Ajuwon, Oguntibeju & Marnewick, 2014).

21 Table 1.3 Reactive oxygen species and reactive nitrogen species (Ajuwon, Marnewick & Davids, 2015; Dhawan, 2014).

ROS are exceptionally reactive species containing oxygen and their production can be triggered by a variety of sources classified as endogenous or exogenous as listed in Table 1.4. ROS are primarily produced by the mitochondria via the mitochondrial electron transport chain, complex I and III being the major production sites. These complexes produce ROS in the mitochondrial matrix in the form of O2•- radical, during energy transduction. Other important production sites for ROS include xanthine oxidase, NADPH oxidase and eNOS uncoupling (Dhawan, 2014).

The ROS have both beneficial and harmful effects depending on the concentration status. When produced in normal or moderate physiological concentrations ROS exert beneficial effects, such as, acting as signalling molecules, protect against environmental pathogens by functioning as mediators of inflammation and they play a role in maintaining vascular tone via cGMP (Dröge, Granner & Boveris, 2002; Li, Horke & Förstermann, 2014; Pourova, Kottova, Voprsalova & Pour, 2010; Valko, Leibfritz, Moncol, Cronin, Mazur & Telser, 2007). However, when the mitochondrial electron transport chain is impaired, primarily due to a pathophysiological condition, excess ROS is generated as a by-product. Excess production of ROS in the biological system often induce oxidative stress which results in the degradation of lipids, membranes, proteins and nucleic acids (Scherz-Shouval & Elazar, 2011).

22 Table 1.4 Sources of pro-oxidants (Ajuwon, Marnewick & Davids, 2015).

Exogenous Endogenous

Neutrophils and macrophages during inflammation

Xanthine oxidase NADPH oxidase eNOS uncoupling

Oxidation of haemoglobin

Enzyme activity including cytochrome P450 system

1.14.3 Antioxidants

Antioxidants play a role in anti-oxidant defence system by regulating the balance between ROS production and their elimination. They can either be endogenous or exogenous produced and further classified as enzymatic or non-enzymatic substances (Table 1.5).

Table 1.5 Classification of antioxidant defence system components (Ajuwon et al., 2015).

Endogenous Exogenous

Non-enzymatic Thiols (glutathione, lipoic acid, N-acetyl

cysteine)

Plant phenols (flavonoids and other polyphenol types)

Metals (copper, manganese, selenium and Zinc)

23 1.14.3.1 Endogenous Antioxidant Enzymes

Endogenous enzymes are localised in different areas, functioning to protect the cells against oxidative damage. These innate antioxidant enzymes can be classified as SOD, catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) (Table 1.5).

O2·- is considered a primary and highly reactive ROS radical produced by the mitochondria during energy transduction. Other sources of O2·- include uncoupled eNOS, xanthine and NADPH oxidase. O2·- radical is converted by the SOD into a less reactive compound, hydrogen peroxide (H2O2). SOD can be classified in to 3 main types; Mitochondrial SOD (MnSOD), cytosolic copper/zinc (Cu/Zn) SOD and extracellular SOD. The extracellular SOD is localised in the interstitial spaces of tissues and extracellular fluids, primarily responsible for the SOD activity in the plasma. They function to dismutate the O2·- radical in various areas to protect the cells from oxidative damage (Pamplona & Costantini, 2011).

H2O2 is further metabolised into H2O and oxygen by the CAT enzyme and also by the glutathione coupled enzyme reaction. The glutathione coupled enzyme reaction consist of GPx, GR and glutathione. Glutathione is the most abundant thiol found in all the cells. In most cells, it exist as reduced glutathione (GSH) and functions to detoxify hydroperoxides. Increased GSH cellular levels may be an indicator of the pathophysiological changes. The glutathione coupled enzyme reaction hydrolyses H2O2 by using the GSH as a substrate and is oxidised to glutathione disulphide (GSSG) by GPx. GSH is in turn regenerated via NADPH oxidation by the GR. This reaction is rendered the most important pathway in H2O2 metabolism in most cells and provides protection against oxidation of lipids (Pamplona & Costantini, 2011).

1.14.3.2 Exogenous Antioxidant Enzymes

Excessive ROS production including other free radicals tend to overwhelm the innate antioxidant enzyme system, as result, additional exogenous antioxidant defence system is often required (Ajuwon, Marnewick & Davids, 2015). Examples of the exogenous antioxidants, often derived from the diet, include vitamin C (ascorbate), E, A and carotenoids (Table 1. 5).

Vitamin C is the most abundant non-enzymatic antioxidant in the cells and is often compared glutathione. Ascorbate opposes accumulation of ROS and free radicals by donating an electron to their outer orbital, forming an ascorbate radical (Asc^-). Asc^- is later converted to its former state by NADH- and NADPH-dependent dehydroascorbate reductase and GSH-dependent dehydroascorbate reductase (Wells & Xu, 1994). Vitamin E and carotene protect the cells

24 against lipid oxidative damage by decreasing the formation of peroxyl to hydroperoxides groups, thus inhibiting the propagation of lipid peroxidation (Brigelius-Flohe & Traber, 1999;

Esterbauer, Schaur & Zollner, 1991; Pamplona & Costantini, 2011). Plant phenolics also play a major role in the exogenous antioxidant defence system by protecting the cells against oxidative damage. Their numerous health effects are mainly attributed to their polyphenolic content (Liu, 2003). These polyphenols are mostly found in teas, fruits, vegetables, spices and have been proven to possess ameliorative effects on the CVD risk factors and cancer (Weng &

Yen, 2012).